Sterility assay by membrane filtration method: Suitability test for 0.1% EDTA eye drops

Introduction
Eye drops edetate disodium (EDTA) at 0.1% are indicated for removal of calcareous concretions in strip keratopathy, due to their calcium-chelating properties. In accordance with Good Preparation Practices (GPP), this hospital preparation (PH) is submitted to final checks, including physico-chemical and microbiological assays. The aim of this work is to validate suitability test for sterility assay of 0.1% EDTA eye drops.

Materials & method
Membrane filtration for sterility assay, the reference method, was carried out according to the European Pharmacopoeia 11th Ed (EP) (2.6.1). This was performed using the SteritestTM Symbio LFH pump (Merck-Millipore) and canisters fitted with a 0.45 µm cellulose ester membrane (Merck-Millipore). The six recommended strains by the EP ; Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans and Aspergillus brasiliensis were tested. Filtration was carried out with several steps (pre-wetting, eye drop filtration, rinsing and contact with the culture media). Inoculums were prepared from Bioball^® SingleShot calibrated to 30 CFU (Biomérieux). Filtration parameters (rinsing speeds and volumes) were set according to the supplier’s recommendations. Culture media used were thioglycolate (TG) for S. aureus, P.aeruginosa and C.sporogenes, and soy casein hydrolysate (SCH) for B.subtilis, C.albicans and A.brasiliensis. A negative control was performed for each medium. After filtration, canisters were incubated at 30-35°C for TG and 20-25°C for SCH. Bacteria and yeast were incubated for a maximum of 3 and 5 days respectively. The suitability test was repeated three times.

Results
The bacteriostatic activity of these eye drops, linked to polymerase inhibition by chelation of magnesium (Mg2+), was inhibited by the rinsing step. Growth of all microorganisms was observed within the allotted time. Identification reports from bacteriology and mycology laboratories isolated monomorphic culture in each canister. At the end of the respective incubation times, limpid negative controls permit to conclude about the absence of microbial contamination.

Discussion - Conclusion
Absence of any inhibitory effect of EDTA 0.1% eye drops on pathogen growth has been demonstrated. The suitability test for 0.1% EDTA eye drops carried out in accordance with the EP is validated and reproducible. This means that the sterility assay for these preparations can be validated. Finally, in routine practice, this method can be used to check for the absence of microbial growth. Thus, this assay enable final validation microbiologically status of this PH.

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